Abstract Colon cancer is the third leading cause of cancer related deaths in United States of America. Chemoresistance (drug resistance) of tumors is the primary reason for the failure of chemotherapy and the major cause of mortality in colon cancer. The molecular mechanisms involved in chemoresistance or approaches to resensitize cancer cells to chemotherapy remain elusive. Recent studies suggest that gut microbiota and microbial metabolites play a crucial role in the development and progression of colon cancer but their role in chemoresistance remains unknown. Towards this goal, we investigated the influence of commensal microbial metabolite, Urolithin A (UroA) on cancer chemotherapy. UroA is a microbial metabolite derived from ellagic acid and ellagitannins, which are major components of berries and pomegranate. Our preliminary data showed that UroA significantly reduced bacteria induced inflammation in macrophages and protected from lipopolysaccharide induced barrier dysfunction by upregulating junctional proteins in colon epithelial cells. More importantly, UroA significantly increased the chemosensitization of colon cancer cells to 5florouracil (5FU) by down regulating drug transporters and modulating epithelial-mesenchymal transition (EMT) pathways. The combination therapy significantly reduced colony formation of colon cancer cells as well as blocked cancer cell migration. By screening several novel structural analogues of UroA, we have identified a potent compound, UAS03, which displayed increased chemosensitizing activities compared to UroA. Based on these observations, we hypothesize that ?UroA and UAS03 act as chemosensitizing adjuvants in 5-FU therapies through regulation of drug transporters and cancer stemness. The goal of the proposal is to determine the effects of microbial derived metabolites (UroA and its analogue UAS03) on colon cancer chemotherapy, especially 5FU resistant cancers. In aim1, we propose to identify the molecular target of UroA using bioactive biotinylated UroA; to determine the molecular mechanisms of UroA and UAS03 mediated chemosensitization of 5FU-resistance (5FUR) colon cancer cells. In aim 2, we will evaluate the therapeutic efficacies of UroA or UAS03 in combination with 5 FU in preclinical colon cancer models. We will utilize both implantable (tumors generated by 5FUR colon cancer cells) and azoxymethane-DSS models. The successful completion of these studies will delineate regulatory mechanisms of microbial metabolite (UroA) mediated chemosensitization and offer better therapeutic options for colon cancer.